The first section of this proposal will focus on developing a better understanding of somatostatin gene expression. We have demonstrated that only 70 bp of DNA are necessary for correct initiation and tissue specific expression of the rat somatostatin gene. Further deletion of sequences leads to a dramatic fall in level of expression. In addition, Montimini, et al. has reported that sequences within this 60 bp fragment confirm cyclic AMP regulation on the somatostatin gene. Our preliminary results support the idea that several protein factors bind to this 70 bp region, and the """"""""context"""""""" (surrounding sequences) of binding play important roles generating their specificity. We will initially carry out an extensive characterization of the binding activities. This will include DNAase footprinting, methylation interference assays, as well as others. Once we have defined some of the physical properties of these factors, we will attempt to demonstrate that specific alterations within the DNA effect both DNA-protein binding and also expression of the cloned (altered) gene in CA-77 cells. It will also be necessary to develop an in vitro assay for somatostatin expression and to analyze the partially purified proteins in this system. Our plan is to ultimately purify the factors which appear to be important for somatostatin gene regulation. The second section of the proposal will attempt to introduce improvements in the synthesis of synthetic genes. We will use the somatostatin cDNA as our model system. Using the sythetic gene, we will attempt to produce quantities of the prohormone large enough that crystals can be obtained and X-ray analysis of these crystals can be initiated.
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